Madhab K. Chattopadhyay

The multifaceted roles of antibiotics and antibiotic resistance in nature

Antibiotics are chemotherapeutic agents, which have been a very powerful tool in the clinical management of bacterial diseases since the 1940s. However, benefits offered by these magic bullets have been substantially lost in subsequent days following the widespread emergence and dissemination of antibiotic-resistant strains. While it is obvious that excessive and imprudent use of antibiotics significantly contributes to the emergence of resistant strains, antibiotic resistance is also observed in natural bacteria of remote places unlikely to be impacted by human intervention. Both antibiotic biosynthetic genes and resistance-conferring genes have been known to evolve billions of years ago, long before clinical use of antibiotics. Hence it appears that antibiotics and antibiotics resistance determinants have some other roles in nature, which often elude our attention because of overemphasis on the therapeutic importance of antibiotics and the crisis imposed by the antibiotic resistance in pathogens. In the natural milieu, antibiotics are often found to be present in sub-inhibitory concentrations acting as signaling molecules supporting the process of quorum sensing and biofilm formation. They also play an important role in the production of virulence factors and influence host–parasite interactions (e.g., phagocytosis, adherence to the target cell, and so on). The evolutionary and ecological aspects of antibiotics and antibiotic resistance in the naturally occurring microbial community are little understood. Therefore, the actual role of antibiotics in nature warrants in-depth investigations. Studies on such an intriguing behavior of the microorganisms promise insight into the intricacies of the microbial physiology and are likely to provide some lead in controlling the emergence and subsequent dissemination of antibiotic resistance. This article highlights some of the recent findings on the role of antibiotics and the genes that confer resistance to antibiotics in nature.

Mechanism of bacterial adaptation to low temperature

Survival of bacteria at low temperatures provokes scientific interest because of several reasons. Investigations in this area promise insight into one of the mysteries of life science —namely, how the machinery of life operates at extreme environments. Knowledge obtained from these studies is likely to be useful in controlling pathogenic bacteria, which survive and thrive in cold-stored food materials. The outcome of these studies may also help us to explore the possibilities of existence of life in distant frozen planets and their satellites.

Increase in Oxidative Stress at Low Temperature in an Antarctic Bacterium

Association between cold stress and oxidative stress was demonstrated by measuring the activity of two antioxidant enzymes and the level of free radicals generated in two batches of cells of an Antarctic bacterium Pseudomonas fluorescens MTCC 667, grown at 22 and 4°C. Increase in oxidative stress in cells grown at low temperature was evidenced by increase in the activity of an enzyme and also in the amount of free radicals generated, in the cold-grown cells. The association between cold stress and oxidative stress demonstrated in this investigation bolsters the concept of interlinked stress response in bacteria.

Antibiotic Resistance of Bacteria

Antibiotic resistance of bacteria and other microorganisms is one of the most serious and grievous challenges of the twenty-first century. The life-saving drugs, which held a great deal of promises during the 1940s to eradicate all the infectious life-threatening diseases in the world, have ceased to work, because of the increasing emergence of microbial strains invulnerable to them. Many of the previously efficacious antibiotics are no longer usable because of widespread occurrence of multiresistant microbial strains. Lately, discovery of new antibiotics is failing to keep pace with the emergence of (multi)resistance of pathogenic and also environmental bacterial strains. Consequently, the prospect of chemotherapy looks bleak. The trepidation that we might be pushed back to a situation analogous to the preantibiotic era, when no chemotherapeutic agent was available to contain and combat deadly bacterial infections, does not appear to be an overblown imagination. Based on this backdrop, this special issue appears to be an aptly undertaken and well-timed endeavour to address this global problem. The articles contributed by investigators from various research laboratories with different scientific backgrounds have not only portrayed the width of the problem but also displayed some silver lining in the management of the looming crisis. Rapid detection of the profile of resistance is essential for timely application of the right antibiotic to a patient. H. Frickmann et al. summarize the efficacy and limitations of various molecular and mass spectrometric methods for the detection of resistance. The omnipresent nature of the resistant organisms is revealed in a number of articles. F. B. Atique and Md. M. R. Khalil report on the occurrence of antibiotic resistance among bacteria (predominantly skin commensal coagulase-negative staphylococci) isolated from allogenic bone samples for grafting, collected from different hospitals of Bangladesh. Food materials are believed to serve as a vehicle for transmission of resistance. This issue is addressed by F. S. Dehkordi et al. who report on the genotype and resistance-profile of Helicobacter pylori isolated from vegetables and salad samples, picked up from groceries and supermarket in a province of Iran. The high similarity in the genotype pattern of the isolates obtained from vegetables and humans indicates transmission. A. B. Florez et al. reveal tetracycline and erythromycin-resistant bacteria and genes conferring resistance to these antibiotics in 10 Spanish and 10 Italian samples of commercial cheese. P. Krupa et al. report on the population structure (based on spa typing) of oxacillin-resistant Staphylococcus aureus isolated from nasal swabs of pigs, collected from two slaughter houses of Poland. Some meat samples bought from the shops were also included into their studies. D. De Vito et al. characterize multidrug-resistant clinical isolates of Salmonella typhimurium for resistance genes in an area of southern Italy by pulsotyping and phage typing. C. Zhang et al. report on the resistant phenotype and genotype of Streptococcus suis serotype 2, isolated from 62 clinically healthy sows and 34 diseased pigs reared in different farms of China. Antibiotic resistance in the nosocomial isolates is a matter of serious concern. F. Lombardi et al. look into the molecular epidemiology of carbapenemase-producing strains of Klebsiella pneumoniae isolated from the surgery unit at a cardiovascular centre of Italy. D. Ojdana et al. demonstrate the ability of an E. coli strain obtained from a hospital of Poland to produce carbapenemase enzymes and also the presence of genes responsible for the production of carbapenemases and other β-lactamases. Extended-spectrum-β-lactamase (ESBL) is a bacterial enzyme having the ability to hydrolyse even the third-generation cephalosporins and aztreonam. Besides Klebsiella pneumoniae some strains of Escherichia coli are also known to produce this enzyme. This is indicated by M. S. Rezai et al. who performed genotyping of ESBL-producing strains of E. coli, obtained from a paediatric hospital of north Iran. The authors also show the association of ESBL-positive E. coli strains with resistance to various other antimicrobials. Occurrence of ESBL-producing Enterobacteriaceae in iceberg lettuce obtained from the retail market of Rochester (US) is described by N. Bhutani et al. A wide spectrum of diseases is caused by the virulent strains of ESBL-positive isolates of E. coli. Regional difference in the prevalence of virulence genes in 432 phenotypically ESBL-positive patient-isolates of E. coli (obtained from the Baltic Sea region) is shown by J. Lillo et al. Keeping in mind the tremendous challenge posed by drug-resistant tuberculosis, a number of relevant articles are included in this collection. The susceptibility profile of M. tuberculosis isolates to various antitubercular antibiotics varies significantly depending on the test system as revealed by Z. Mei et al. They have also shown that changes in bacterial susceptibility are further caused by mixed infection with particular genotypes of M. tuberculosis strains. Resistance-profile of 100 strains of M. tuberculosis, isolated from patients in northeast Iran, is reported by A. T. Sani et al. Occurrence of nontuberculosis Mycobacterium, in 25 out of 125 patients (20%) surveyed, underscores the need of proper diagnosis before the onset of chemotherapy. Discovery of new drugs and strategies to circumvent antibiotic resistance is the need of the hour to contain the problem. N. Jafari et al. report on the isolation of an antibiotic-producing strain of a soil Actinomycetes belonging to the genus Pseudonocardia. The antibacterial compound produced by it is effective against Staphylococcus aureus. They have also purified and partially characterized this compound. R. D. Wojtyczka et al. demonstrate high antibacterial activity of two new quinoline derivatives of a structure of 3-thioacyl 1-methyl 4-arylaminoquinolinium salts against some nosocomial strains of staphylococci in both planktonic and biofilm form. In view of the widespread nature of the problem caused by inefficacy of the antibiotics produced by fermentation and chemical synthesis, it is necessary to tap alternative sources (e.g., plant kingdom) for novel antibiotics. P. Del Serrone et al. demonstrate antibacterial activity of Neem seed oil (Azadirachta indica A. Juss.) against enteropathogenic strains of E. coli and indicate that some of the ciprofloxacin-resistant isolates lost their virulence following treatment with Neem seed oil. Antimicrobial peptides are considered potential candidates for the management of multidrug-resistant infections. M. Singh and K. Mukhopadhyay evaluate the antimicrobial potential of an anti-inflammatory neuropeptide whereas C. Chen et al. report on the efficacy of recombinant lysostaphin against methicillin-resistant S. aureus (MRSA) in a mouse model. Widespread use of carbapenems is associated with emergence of resistance. The polymyxin antibiotic colistin is not used at present because of its nephrotoxicity. H.-J. Tang et al., however, demonstrate the efficacy of a combination of colistin and imipenem against carbapenem-resistant Klebsiella pneumoniae. Bacteriophages could be suitable alternatives for antibiotics, which currently have lost efficacy because of the emergence of resistant strains. N. Shivshetty et al. demonstrate the potential of a bacteriophage isolated from sewage to protect diabetic mice against Pseudomonas aeruginosa-induced bacteremia. Reversal of bacterial resistance to antibiotics is essential to restore the efficacy of the existing antimicrobials. C. Santiago et al. claim to achieve an increase in susceptibility of a MRSA strain to ampicillin when it was combined with a plant extract. A number of computerized models have been developed during the recent past to assist the physicians with the necessary information to enable prescription of the right antibiotic in the right moment. M. Rodriguez-Maresca et al. report on the efficacy of a new electronic device based on laboratory data on the most probable susceptibility profile of pathogens responsible for infections and also on local epidemiology.

Use of antibiotics as feed additives: a burning question

Antibiotics are chemotherapeutic agents used for the clinical management of infectious diseases in humans, plants and animals. However a sizeable fraction of antibiotics produced every year all over the world is used for non-therapeutic purposes. In US alone, about 24.6 million pounds of antibiotics are used in animal agriculture annually and a substantial portion of this is used as growth promoters and not for the treatment of infections (Oliver et al., 2011). According to a recent report, out of 13 million kg of antibiotics administered to animals in 2010, the major portion was meant for promoting the growth of the livestock (Spellberg et al., 2013). The ability of low doses antibiotics to promote growth of animals and birds was discovered serendipitously in the 1940s (Gustafson and Bowen, 1997). Subsequently, it was widely exploited and by this time, addition of antibiotics to the animal feed to stimulate growth has turned into a global practice. The basis of growth-promoting effect of antibiotics is not clearly known. It is postulated that microorganisms present in the animal feed consume a considerable portion of nutrients in the feed. They also inhibit absorption from the intestine and produce toxins having adverse effect on the health of the animals. The growth-promoting effect of antibiotics might stem from their ability to suppress these harmful organisms. It is also suggested that animals reared in the unhygienic environments always bear some latent infections, which trigger a cascade of events in their immune system. Cytokines produced in the process lead to the release of some catabolic hormones which cause wastage of muscles. Antibiotics relieve the animals of the need to produce cytokines by suppressing the causative agents of infections.

A branched chain fatty acid promotes cold adaptation in bacteria

Bacterial strains that can survive extreme cold do so by adopting special strategies. At low environmental temperature, the fluidity of bacterial cell-membranes decreases and maintenance of an optimum membrane fluidity becomes crucial for survival. Incorporation of lower-melting point fatty acids (unsaturated, short chain and branched chain fatty acids) into lipids exerts a fluidizing effect on the membrane. These changes are known as the homeoviscous adaptation of membrane fluidity (Suutari and Laakso 1994). Among branched chain fatty acids, synthesis of anteiso fatty acids increases in preference to the synthesis of iso fatty acids. This is a common change induced by a decrease in temperature. Branching occurs from the penultimate carbon atom furthest from the functional group in an anteiso fatty acid whereas branching occurs from the furthest carbon atom in an iso fatty acid. There is accumulating evidence that anteiso fatty acids play an important role in cold adaptation of bacteria. An anteiso fatty acid (a–C15:0) was found to be the major component in the fatty acid profile of one Gram-positive and one Gram-negative Antarctic psychrotroph, grown at low temperature (Chattopadhyay and Jagannadham 2001). Listeria monocytogenes is a food-borne pathogen that grows at refrigeration temperatures. In two strains of L. monocytogenes, a predominance of a–C15:0 was found in the fatty acid profile when cells were grown at 5°C. Two cold-sensitive mutants of L. monocytogenes were found to be deficient in the synthesis of a–C15:0 and also in the synthesis of another branched chain fatty acid, a–C17:0. It is known that a switchover in the synthesis from iso to anteiso fatty acids in bacteria depends on selection of the proper primer. The primer for anteiso odd-numbered fatty acids is the CoA ester of 2-methylbutyric acid. It is derived from isoleucine. It was postulated that the cold-sensitivity of the mutants might stem from their inability to produce 2-methylbutyryl CoA. By adding 2-methylbutyric acid to the culture it was possible to restore both the ability of the mutants to grow at low temperature and their level of a–C15:0 and a–C17:0 to that found in the parent strain (Annous et al 1997). A question still remained regarding the exact role of these two components in membrane fluidity. Recently the gap has been bridged by measuring the membrane fluidity of one of the mutants and the parent strain with the help of electron paramagnetic resonance (EPR). The membrane of the mutant cell was found to be less fluid than the membrane of the parent strain. When the mutant was grown in the presence of 2-methylbutyric acid, its membrane fluidity was found to be restored to a level comparable to that of the parent (Jones et al 2002). It is evident that these two-branched chain fatty acids significantly improve membrane fluidity of L. monocytogenes at low temperature and hence contribute to cold adaptation in this organism. Studies involving Antarctic bacteria indicate that a–C15:0 may play a beneficial role in general in bacterial cold adaptation. Annous et al (1997) showed that the amount of the major component of the fatty acid profile of L. monocytogenes at low temperature (a–C15:0) was slightly enhanced when glycine betaine was present in the growth medium. The role of a–C15:0 in membrane fluidity supports the hypothesis that glycine betaine acts as a cryoprotectant by virtue of its ability to enhance the biosynthesis of membrane fluidizing fatty acids.

Vesicles-mediated resistance to antibiotics in bacteria.

During the past few decades, antibiotic-resistance of bacteria has assumed the proportion of a global crisis. The discovery of new antibiotics is not matching the rate of emergence of resistant strains thus narrowing the scope of chemotherapy. Notwithstanding the fact that emergence of resistance is facilitated by the use of antibiotics (Levy, 2002) exposure to antibiotics is not a pre-requisite for it. Antibiotic-resistance is detected even in bacteria occurring in places detached from the human civilization for millions of years (Bhullar et al., 2012). The various mechanisms responsible for antibiotic-resistance of bacteria were discussed in this journal some time back (Lin et al., 2015). Outer Membrane Vesicles (OMVs) are spherical bag-like structures (20–300 nm) released predominantly by gram-negative bacteria in the outer environment. They help the producer cells in communication with other cells, secretion, pathogenesis, acquisition of nutrients, and self-defense (Kulkarni and Jagannadham, 2014). Recent evidences indicate that they protect bacteria not only from phages and various environmental stress factors but also some antibiotics. The nature of vesicle-mediated antibiotic-resistance, known so far, is dealt with in this article.

Metabolism in bacteria at low temperature: A recent report

The adaptability of bacteria to extreme cold environments has been demonstrated from time to time by various investigators. Metabolic activity of bacteria at subzero temperatures is also evidenced. Recent studies indicate that bacteria continue both catabolic and anabolic activities at subzero temperatures. Implications of these findings are discussed.

Cryotolerance in bacteria: interlink with adaptation to other stress factors

Investigations on bacterial adaptation to extreme low temperatures provide insight into the intricacies of the cellular machinery, offer clues in exploring the possibility of the existence of life in remote planets and identify molecules of biotechnological importance (e.g. cold-active protease and thermolabile alkaline phosphatase). A careful look into the plethora of information that has been generated during the past few decades reveals the role of some molecules in linking bacterial cryotolerance to some other types of stress adaptation.The heat shock protein (HSP) ClpB, which is known to be essential for acquired thermotolerance in cyanobacteria and eukaryotes, was found to be strongly induced during moderate cold stress in the cyanobacterial strain Synechococcus PCC7942. Both growth and photosynthesis at low temperature were adversely affected in a clpB deletion mutant of the strain [1xInduction of the heat shock protein ClpB affects cold acclimation in the cyanobacterium Synechococcus sp. strain PCC 7942. Porankiewicz, J. and Clarke, A.K. J. Bacteriol. 1997; 179: 5111–5117PubMedSee all References[1]. The role of HtpG, a homologue of Hsp90, in the survival of the cyanobacterial strain Synechococcus PCC7942 under heat stress, and also in the acclimation of the same strain to low temperature and oxidative stress, was demonstrated [2xRole for the cyanobacterial HtpG in protection from oxidative stress. Hossain, M.M. and Nakamoto, H. Curr. Microbiol. 2003; 46: 70–76Crossref | PubMed | Scopus (37)See all References[2]. It seems that it was the chaperoning properties of the HSPs that served as a link between the adaptation to low and high temperatures, two stress factors that are diametrically opposite in nature. The postulation is bolstered by fact that ClpB is actually a bacterial homologue of the molecular chaperone Hsp104, which is known to reverse protein aggregation by rescuing polypeptide chains from aggregates and facilitating their refolding [3xThe molecular chaperone Hsp104 – a molecular machine for protein disaggregation. Bosl, B. et al. J. Struct. Biol. 2006; 156: 139–148Crossref | PubMed | Scopus (73)See all References[3]. It is also well documented that some compounds called chemical chaperones (e.g. glycine betaine and proline), which are known to stabilize the native conformation of cellular proteins, were found to have a protective role against cold stress, salt stress and thermal stress in bacteria [4xComparative analysis of naturally occurring L-amino acid osmolytes and their D-isomers on protection of Escherichia coli against environmental stresses. Shahjee, H.M. et al. J. Biosci. 2002; 27: 515–520Crossref | PubMedSee all References, 5xThe cryoprotective effects of glycine betaine on bacteria. Chattopadhyay, M.K. Trends Microbiol. 2002; 10: 311Abstract | Full Text | Full Text PDFSee all References, 6xThe chemical chaperone proline relieves the thermosensitivity of a dnaK deletion mutant at 42 °C. Chattopadhyay, M.K. et al. J. Bacteriol. 2004; 186: 8149–8152Crossref | PubMed | Scopus (42)See all References]. Treating the bacterial cells with two different groups of antibiotics (which all acted on ribosomes) that were found to mimic the upshift and downshift of temperature in Escherichia coli led to the synthesis of HSPs and cold shock proteins (CSPs), respectively [7xRibosomes as sensors of heat and cold shock in Escherichia coli. VanBogelen, R.A. and Neidhardt, F.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5589–5593Crossref | PubMed | Scopus (319)See all References[7]. Subsequently, the syntheses of some HSPs and CSPs were found to be induced in E. coli under high hydrostatic pressure [8xStress response of Escherichia coli to elevated hydrostatic pressure. Welch, T.J. et al. J. Bacteriol. 1993; 175: 7170–7177PubMedSee all References[8]. Hence, bacterial ribosomes seem to have the role of intracellular sensors, which integrate the adaptation of the organism to high temperatures, low temperatures and high pressure. An interlink between the cold tolerance and acid tolerance of Lactobacillus delbrueckii has been evidenced very recently by the enhanced freeze-tolerance of some cells that were acidified at pH 5.25 for 30 min at the end of fermentation [9xAcid adaptation of Lactobacillus delbrueckii subsp. bulgaricus induces physiological responses at membrane and cytosolic levels that improves cryotolerance. Streit, F. et al. J. Appl. Microbiol. 2008; 105: 1071–1080Crossref | PubMed | Scopus (31)See all References[9].The multifaceted role of the stress combatants discussed here indicates that there might be some commonality in the damaging effects of the physicochemical factors that inflict stress on bacteria. Further investigations on this aspect are likely to offer some more clues to the adaptability of bacterial cells to extreme environments. The ability of chaperones, both protein-based and chemical, to confer cold tolerance in bacteria might be maneuvered to improve the growth rate at low temperatures of some mesophilic bacteria [10xChaperonins govern growth of Escherichia coli at low temperatures. Ferrer, M. et al. Nat. Biotechnol. 2003; 21: 1266–1267Crossref | PubMed | Scopus (115)See all References[10], which can be subsequently used for bioremediation in harsh environments. The phenomenon of interlinked stress response thus provides a multitude of opportunities for both basic and applied research.

The link between bacterial radiation resistance and cold adaptation.

Mechanisms of cold adaptation in bacteria remain by and large ill-defined. Investigations on psychrotrophic strains have provided some clues (Chattopadhyay and Jagannadham 2001). Recent reports reveal that there are specific factors which promote survival of bacteria not only at low temperature, but also under other types of stress conditions. Thus they provide a link between different types of stress adaptations. For example, monounsaturated fatty acids were found to be important for the growth of a deep sea bacterium in cold, high pressure zones of ocean depth (Allen et al 1999). Substantial improvement in survival of an Escherichia coli strain was noticed during frozen storage following the inductive synthesis of some heat shock proteins which are believed to protect bacteria from thermal stress (Chow and Tung 1998). Recently it has been demonstrated that specific genes are over-expressed during both oxidative stress and cold stress in E. coli (Smirnova et al 2001). The work highlighted here provides a possible link between cold adaptation and radiation resistance (Mangoli et al 2001).